Mutations might stabilize certain conformations of an enzyme and can, for example, result in a higher catalytic activity, a higher affinity for the substrate, a different substrate specificity or a higher thermostability of the enzyme. In this context it can also be argued that evolution modifies the energy landscape to find an optimal compromise between all these parameters.
Allosteric effectors or covalent modifications might shift a pre-existing equilibrium between an active and an inactive enzyme conformation thereby stabilizing either the active or an inactive conformation.
Changes in the microenvironment are known to directly influence enzymatic activity. The most obvious influence might be the temperature which is known to determine the height of the barriers of the energy landscape.
An external stretching force can be seen as some sort of “artificial allosteric effector” and has a very well-characterized influence on the energy landscape of biological systems. Forces play an important but largely unexplored role in many biological processes

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We try to understand the influence of these factors on the energy landscape with single molecule experiments. Ultimately, single molecule experiments can reveal how a specific modification influences the energy landscape and shifts the equilibrium between different conformations. Our approach is a combination of single molecule fluorescence and force spectroscopy with molecular biology, biohybrid chemistry and organic chemistry to prepare and modify our enzymes and enzyme substrates.

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Enzyme Function and Regulation
Single molecule experiments with lipase B from Candida antarctica (CalB) have shown that this enzyme has large fluctuations in the turnover rate and more importantly that it is only active for 3 % of the overall measurement time. Our current experiments aim at finding ways to increase the overall activity of CalB (with mutations, covalent modifications, buffer conditions, etc.) and to analyze the effect of these modifications at the level of individual enzymes.

We use a fluorescence based approach to detect enzymatic activity. Using a so-called fluorogenic substrate, which is cleaved by to enzyme to yield a fluorescent product molecule, we are able to detect individual enzymatic turnover reactions with a confocal fluorescence microscope. Surface immobilization of the enzyme ensures that the same enzyme can be monitored for extended periods of time. Subsequent data analysis of the times between individual turnover reactions ultimately yields the information how a specific modification alters the energy landscape and how the energy landscape determines the catalytic activity.

Collaborations:
Alan Rowan
Hans Engelkamp, Peter Christianen, Jan C. Maan
Jan van Hest
Johan Hofkens
Chun Biu Li
Klaus Schulten

Related publications:

1. Blank, K., G. De Cremer, and J. Hofkens. (2009). Fluorescence-based analysis of enzymes at the single-molecule level. Biotechnol J 4:465-479.
2. Engelkamp, H., N. S. Hatzakis, J. Hofkens, F. C. De Schryver, R. J. Nolte, and A. E. Rowan. (2006). Do enzymes sleep and work? ChemCommun (Camb):935-940.
3. Flomenbom, O., K. Velonia, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. Nolte, M. Van der Auweraer, F. C. de Schryver, and J. Klafter. (2005). Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules. Proc Natl Acad Sci USA 102:2368-2372.
4. Velonia, K., O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. Nolte, and F. C. de Schryver. (2005). Single-enzyme kinetics of CALB-catalyzed hydrolysis. Angew Chem Int Ed Engl 44:560-564.

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Evolution of Promiscuous Enzymes
The most prominent evolution theory suggests that the evolution of a new enzymatic activity is linked to a different enzyme conformation which is stabilized on the energy landscape during the evolution process. In this context, a promiscuous enzyme would exist in at least two conformations: one conformation catalyzes the natural reaction while a second conformation is responsible for the promiscuous activity.
We are performing directed evolution with promiscuous enzymes with the goal of increasing the promiscuous activity. Different variants from different steps of the evolution process will then be analyzed with two different fluorogenic substrates (one for the natural activity and the other for the promiscuous activity) at the single molecule level. Single molecule experiments are the ultimate approach to record the sequence of turnovers of both substrates and to tell us how the promiscuous activity increases and how it is determined by different enzyme conformations.

Bachelor and Master projects available

Collaborations:
Floris van Delft,Floris Rutjes
Michael Ryckelynck, Andrew Griffiths

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Enzyme Substrates
Fluorogenic substrates are a crucial component of our single enzyme experiments. In an ideal case every single turnover reaction generates one fluorescent dye molecule which can be detected with a good signal to noise ratio. Many of the currently used substrates, however, do not provide this 1:1 stoichiometry. Furthermore, the application of these substrates is limited by autohydrolysis and low solubility. We are currently developing improved substrates for lipases, proteases and phosphoesterases.

Bachelor and Master projects available

Collaborations:
Alan Rowan
Floris van Delft, Floris Rutjes

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Enzyme immobilization
Single enzyme experiments require the immobilization of the enzyme in the confocal volume. It is crucial that the immobilization procedure does not introduce heterogeneities in the enzyme population. We are currently developing and comparing a variety of immobilization methods based on adsorption, gel encapsulation, coupling to polymer networks and site-specific coupling to poly(ethylene glycol) spacers.

Bachelor and Master projects available

Collaborations:
Alan Rowan
Hans Engelkamp, Peter Christianen, Jan C. Maan
Jan van Hest

Related publications:

1. J. L. Zimmermann, T. Nicolaus, G. Neuert, K. Blank (2010) Thiol-based, site-specific and covalent immobilization of biomolecules for single molecule experiments. Nature Protocols 5:975-985, doi:10.1038/nprot.2010.49
2. Blank, K., J. Morfill, and H. E. Gaub. (2006). Site-specific immobilization of genetically engineered variants of Candida antarctica lipase B. Chembiochem 7:1349-1351.
3. Schoffelen, S., M. H. Lambermon, M. B. van Eldijk, and J. C. van Hest. (2008). Site-specific modification of Candida antarctica lipase B via residue-specific incorporation of a non-canonical amino acid. BioconjugChem 19:1127-1131.

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Integration of Single Molecule Techniques
We have recently shown that the activity of CalB can be manipulated by stretching the enzyme with an atomic force microscope (AFM). Although CalB is not naturally subjected to stretching forces, there are a variety of biological processes which are mechanically regulated. Continuing our studies with CalB, we are now developing an AFM-fluoresence set-up which will allow us to monitor enzymatic activity at various constant stretching forces in order to understand the process of force activation. In addition to the combination of single molecule fluorescence with force spectroscopy we also aim to combine single molecule fluorescence with electronic detection of individual enzymatic reaction events. This combination will potentially allow us to use a broader range of (naturally) charged substrates and therefore significantly extend the range of enzymes which can be studied. No artificial fluorescent reporter molecules will be required anymore.

Collaborations:
Alan Rowan
Peter Christianen, Hans Engelkamp, Jan C. Maan
Hermann Gaub
Ethan Minot

Related publications:

1. Gumpp, H., E. M. Puchner, J. L. Zimmermann, U. Gerland, H. E. Gaub, and K. Blank. 2009. Triggering enzymatic activity with force. Nano Lett 9:3290-3295.

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